Campylobacter Colitis: Histological Immuno- Histochemical and Ultrastructural Findings
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Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from Gut, 1985, 26, 945-951 Campylobacter colitis: histological immuno- histochemical and ultrastructural findings J P VAN SPREEUWEL, G C DUURSMA, C J L M MEIJER, R BAX, P C M ROSEKRANS, AND J LINDEMAN From the Department ofInternal Medicine, Division of Gastroenterology, St. Antonius Ziekenhuis, Nieuwegein, Department ofMicrobiology and Pathology, Stichting Samenwerking Delftse Ziekenhuizen, Delft, Department ofInternal Medicine, St. Elizabeth Ziekenhuis, Leiderdorp, and the Department of Pathology, Vrije Universiteit, Amsterdam, The Netherlands SUMMARY The colonic biopsy specimens of 22 patients with colitis and positive stool cultures for Campylobacter jejuni were studied in order to obtain histological and immunohistochemical criteria to differentiate Campylobacter colitis from chronic inflammatory bowel disease. In addition we tried to identify Campylobacter inclusions by means of immunohistochemistry and electron microscopy as evidence for invasion of the colonic mucosa. The results show that the majority of patients with Campylobacter colitis have the histological picture of acute infectious colitis with increased numbers of IgA and IgM containing plasma cells in the colonic mucosa in contrast with patients with active chronic inflammatory bowel disease who show increases of IgA and IgG (ulcerative colitis) or IgA-, IgM and IgG containing plasma cells (M Crohn) in their colonic biopsies. The results of immunohistochemical stainings with Campylobacter antiserum show invasion of Campylobacter in the colonic mucosa. These findings were confirmed ultrastructurally. http://gut.bmj.com/ In the past decade Campylobacter has emerged be indistinguishable from ulcerative colitis. Histo- from obscurity to become an important human logical examination of rectal biopsies has ranged pathogen. Provided that proper techniques are from normal to inflammatory changes suggestive of applied Campylobacter fetus subspecies jejuni can acute infectious colitis or ulcerative colitis or be isolated from the stools of patients with diarrhoea Crohn's disease.61' In the present study we have on September 30, 2021 by guest. Protected copyright. as often as Salmonella.1 2 In the laboratory for investigated the colonic biopsies of 22 patients with microbiology of the SSDZ, Delft, Campylobacter diarrhoea and stool cultures positive for Campylo- jejuni was isolated as the causative organism in 41% bacter jejuni. The aim of our study was to determine of 732 consecutive positive stool cultures of patients the histological features and the number of immuno- with diarrhoea. Campylobacter may cause a spec- globulin containing cells in colonic biopsies of trum of intestinal disease ranging from acute gas- patients with Campylobacter colitis and to identify troenteritis to toxic megacolon, lymphadenitis Campylobacter inclusions immunohistochemically. mesenterialis, and even appendicitis and cholecysti- tis.35 Usually infection with this organism results in Methods acute gastroenteritis with fever and frequent loss of often bloody stools. The disease resolves spon- PATIENTS taneously within one week but in 20% it runs a more One hundred and twelve colonic biopsies from 22 prolonged course or relapses resembling chronic patients with positive stool cultures for Campylobac- inflammatory bowel disease.1 Endoscopically it may ter jejuni were examined. One patient had been suffering from chronic ulcerative colitis for many years whereas one patient had previously suffered Address for correspondence: Dr J P van Spreeuwel, Department of Internal from non-specific proctitis. The others had no Medicine, Division of Gastroenterology, Ziekenhuis St. Annadal, St. Annadal 1, 6214 PA Maastricht, The Netherlands. coexisting gastrointestinal disease. Diarrhoea with Received for publication 1 November 1984 crampy abdominal pain was the presenting com- 945 Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from 946 van Spreeuwel, Duursma, Meijer, Bax, Rosekrans, and Lindeman plaint in 15 patients, 13 of them had noticed blood in 0-5 mm stretched muscularis mucosae. The latter is their stools, and fever was an accompanying symp- comparable with the 'mucosal tissue unit' described tom in seven of these 15 patients. Nausea was by Brandzaeg and Baklien.14 Moreover, immuno- recorded frequently. Six patients merely complained histochemistry was carried out with Campylobacter of loosing blood with their stools. One patient antiserum using both direct immunoperoxidase and presented with abdominal pain, nausea, and vomi- immunofluorescence techniques. The antiserum was tus. The numbers of immunoglobulin containing raised in a rabbit that was vaccinated intravenously cells in colonic biopsies of patients with Campylo- with a Campylobacter fetus subspecies jejuni bacter colitis were compared with those of 10 suspension containing microorganisms of serotype healthy controls, 10 patients with active Crohn's LAU1'5 that were killed in 1-5% formalin. Anti- disease of the colon and 10 patients with active serum titres were determined by agglutination and ulcerative colitis. All patients with chronic inflam- indirect immunofluorescence methods. After matory bowel disease had histologically abnormal several absorption steps with Salmonella g, Shigella colonic biopsies. boidii, Shigella sonnei, Proteus vulgaris and Escher- Stools were cultured for Salmonella, Shigella, ichia coli the antiserum showed no cross reactivity Yersinia and Campylobacter routinely. Campylo- with Salmonella, Shigella, or Escherichia coli. The bacter was cultured on a selective medium: blood antiserum gave a positive immunofluorescence with agar plates containing Campylobacter Growth Sup- 37 out of 38 different Campylobacter LAU sero- plement and Campylobacter Selective Supplement types; representing all known serotypes at that time (Oxoid LTD Basingstoke England nos. SR 84 and in the Netherlands. Immunofluorescence was SR 85). Inoculated plates were incubated during 48 achieved by removing paraffin with xylol and hours at 42°C in an atmosphere containing 5-15% alcohol, preincubation with normal swine serum 02 and 5-8% CO2 by using the GasPak of BBL 1:10, incubation with rabbit Campylobacter anti- without a catlyst in a jar. serum 1:40, once again incubation with normal Biopsies were fixed immediately either in a swine serum 1:10 and incubation with swine anti- formalin sublimate mixture for four to six hours for rabbit FITC. Sections stained with preimmune routine histology and immunohistochemical studies serum from the same rabbit served as controls. or in cacodylate buffered glutaraldehyde for ultra Colonic biopsies from eight patients with Crohn's structural studies. Tissue samples were embedded in disease of the colon and nine patients with ulcerative paraffin wax, cut in sections 4,um thick and mounted colitis stained with Campylobacter antiserum served http://gut.bmj.com/ on glass slides. Besides histological stains including as controls as well and were all negative. In addition haematoxylin and eosin and periodic acid Schiff, in three patients in whom immunohistochemistry biopsies were stained specifically for IgA, IgM, and with Campylobacter antiserum had given positive IgG heavy chains using an indirect immunoperox- results ultrastructural examination of the biopsies idase technique.12 Appropriate controls were done was done using a Zeiss EM 109 electron microscope. according to Sternberger.13 Rabbit antisera against Statistical analysis was carried out according to IgA, IgM, and IgG heavy chains were purchased the Student's t test, p<005 was considered signifi- from Dakopatts (Denmark). Their specificity was cant. on September 30, 2021 by guest. Protected copyright. confirmed by immunoelectrophoresis, immuno- fluorescence and immunoperoxidase stainings on Results bone marrow preparations monoclonal for IgA, IgM, or IgG. Horseradish peroxidase labelled goat The results are summarised in the Table. antirabbit Ig was obtained from Miles (Yedah, Israel). The IgA, IgM, and IgG stained sections HISTOLOGICAL FINDINGS were used for morphometrical analysis. They were In 19 patients the colonic biopsies showed a histolo- photographed with a standard magnification (100, 8 gical picture consistent with acute infectious colitis. x) and projected on a graphic tablet interfaced to a In the patient with longstanding ulcerative colitis computer (Ibas I Kontron Munchen). Every photo- with superimposed Campylobacter enterocolitis the graph contained approximately 1 mm mucosa at full histological features of ulcerative colitis dominated thickness. The lamina propria area was limited by with marked distortion of crypt architecture. In two two lines perpendicular to the muscularis mucosae patients histological examination revealed no abnor- and this area was measured per mm muscularis malities. Based on our findings in patients in whom mucosae length. The number of immunoglobulin follow up biopsies were obtained, and the time lapse containing cells was measured in two consecutive between the onset of bloody diarrhoea and the sections in approximately the same area and ex- biopsy we believe the histological changes of Cam- pressed both per 0 1 mm2 lamina propria and per pylobacter colitis can be divided in three stages. Gut: first published as 10.1136/gut.26.9.945 on 1 September 1985. Downloaded from Campylobacter colitis: histological immunohistochemical and ultrastructural findings 947 Table Histological and immunohistochemical